Growth factor signaling plays a critical role in normal development and cancer progression, yet little is known about the signaling pathways utilized during prostate development, and how they may be perturbed during prostate carcinogenesis. Based on our previous studies of the role of the homeobox gene Nkx3.l in prostate development and cancer progression, we are pursuing two parallel and complementary approaches for investigating these signaling pathways. First, we are performing a general unbiased screen to identify prostate inducing factors, based on their expression patterns in the embryonic urogenital sinus and on their activity in a novel in vitro explant culture assay for prostate induction that we have established. This assay is greatly facilitated by our generation of Nkx3.l-1acZ knock-in mice, which permit the use of beta-galactosidase staining as a robust and early marker of prostatic epithelial differentiation. Secondly, we are pursuing a candidate pathway approach, focusing on signaling by the transforming growth factor-beta (TGF-b) pathway, which has been implicated in many aspects of development and carcinogenesis. For this purpose, we will perform a functional analysis of Smad2, which encodes an essential intracellular signal transducing molecule that is a primary mediator of activin and TGF-b signals. In particular, we have successfully generated mice expressing Cre recombinase that should allow prostate-specific gene targeting of Smad2. Therefore, we will pursue two broadly based specific aims that integrate general analyses of prostate formation with a more focused investigation of TGF-b signaling: I) Investigation of mouse prostate induction and candidate inducing factors by examining the expression of putative signaling factors during prostate induction, utilizing an explant culture assay system to identify prostate inducing activities, and analyzing the prostatic phenotype of mice mutant for these inducing factors; and II) Functional analysis of TGF-b signaling in prostate development and cancer by investigating Smad2 activation in normal prostate development and in mouse models of prostate cancer, examining male urogenital development in mice mutant for activin receptors, and analyzing the consequences of Smad2 loss-of-function during prostate development and cancer progression by tissue-specific gene targeting. Our studies should identify critical growth factor pathways for prostate induction and differentiation, and should help elucidate the role of TGF-b signaling in normal prostate development as well as carcinogenesis.